EP0309862B1 - Stilbene synthase gene - Google Patents

Description

The present invention relates to the gene for stilbene synthase isolated from plants and its use for transforming vectors, host organisms and plants and for producing plants which have increased resistance to pests.

The term stilbene describes a group of chemical substances that occur in plants and contain the stilbene framework (trans-1,2-diphenylethylene) as a common basic structure. This basic structure can be supplemented by adding further groups. Two important stilbenes are the 3,5-dihydroxy-stilbene (Pinosylvin) and the 3,3 ′, 5-trihydroxy-stilbene (resveratrol).

Stilbenes were found in certain trees (angiosperms, gymnosperms), but also in some herbaceous plants (in species from the Myrtaceae, Vitaceae and Leguminosae families). Stilbenes are toxic to pests, in particular for fungi, bacteria and insects and are suitable to ward off these pests. Their greatest biological importance is also seen in this function. Particularly in herbaceous plants, it is often the case that stilbenes are only present in very low concentrations in healthy tissue, but that a very large amount of stilbenes is newly formed at the site of infection after infection or wounding. This increased concentration correlates with the increased resistance of the plants that stilbene can synthesize to the pests. The ability to synthesize these substances is seen as an important defense mechanism. Unfortunately, only a few useful plants have the ability to form stilbenes or to produce them to a degree that gives them sufficient resistance to pests.

The key to the formation of all style levels is the synthesis of the basic framework. So far, essentially two types of enzymes have been described (resveratrol synthase and pinosylvine synthase), both of which are referred to as stilbene synthases, since they both synthesize the stilbene backbone. Resveratrol synthase from peanut (Arachis hypogea) has been best characterized so far; the properties of this enzyme are largely known (Schöppner and Kindl, 1984). Both Pinosylvin and Resveratrol, the simplest stilbenes, have a generally toxic, preferably microbicidal, in particular fungistatic, effect on infecting harmful organisms. Stilbene synthases use malonyl-CoA and cinnamoyl-CoA or coumaroyl-CoA as substrates, i.e. substances that occur in every plant because they are also used in the biosynthesis of other important plant ingredients (e.g. flavonoids, flower pigments). On the subject of stilbene and stilbene synthase, reference can be made to the following literature: Hart, JH (1981) Annu. Rev. Phytopathology 19 , 437-458; Hart, JH, Shrimpton, DM (1979) Phytophathology 69 , 1138-1143; Kindl, H. (1985) in: Biosynthesis and Biodegradation of Wood Components. Ed. Higuchi, T., Academic Press, Inc., pp. 349-377 and Schöppner, A .; Kindl, H. (1984) J. Biol. Chem. 259 , 6806-6811.

A large part of the world crop of crops is constantly destroyed by pests (in 1967 the loss of potential harvest was 35%; see Chemistry of Pesticides, edited by K.H. Büchel, John Wiley & Sons, New York, 1983, page 6). There is therefore an urgent need to research and utilize all possibilities which are suitable for reducing or preventing pest infestation in crop plants.

The new gene for stilbene synthase ("stilbene synthase gene") has now been isolated, which can be incorporated into the genetic material (the genome) of plants which do not produce a stilbene or only inadequately stilbene, which causes increased resistance of these plants to pests can.

The stilbene synthesis genes according to the invention correspond to the stilbene synthase gene which is contained in the plasmid pGS 828.1 and include the DNA sequences derived therefrom with stilbene synthase activity.

Stilbene synthase gene should be understood to mean any nucleic acid (DNA) which, after its transcription in RNA and translation in protein (in a suitable environment), causes the formation of an enzyme which has the properties of a stilbene synthase (enzymatic synthesis of stilbene in a suitable environment) has, wherein this nucleic acid is isolated from its natural environment or is integrated into a vector or is contained in a prokaryotic or eukaryotic DNA as "foreign" DNA or as "additional" DNA.

If the stilbene synthase gene still contains DNA sequences at its beginning and / or end which do not or not significantly hinder the function of the gene, the term “gene unit” is also used below. These gene units arise, e.g. by cutting out with restriction enzymes (e.g. partial cleavage with EcoR I and Hind III), since there are no interfaces for conventional restriction enzymes exactly at the beginning and at the end of the gene.

The stilbene synthase gene (or the gene unit) can be in the form as it is contained in the genome of plants ("genomic" form, including non-stilbene synthase-coding and / or non-regulatory sequences (such as introns) or in one Form that corresponds to the cDNA ("copy" DNA) that can be obtained via mRNA with the aid of reverse transcriptase / polymerase (and no longer contains introns).

In the stilbene synthase gene according to the invention or the gene unit), DNA segments can be replaced by other DNA segments which have essentially the same effect. It can also carry those DNA sequences at the ends that are adapted to the handling of the gene (or the gene unit) in each case (e.g. "linker").

In the present context, “foreign” DNA is to be understood as DNA that does not occur naturally in a specific prokaryotic or eukaryotic genome, but is only incorporated into this genome by human intervention. "Additional" DNA should be DNA that occurs naturally in the respective prokaryotic or eukaryotic genome, but is added to this genome by human intervention. The "foreign" DNA or "additional" DNA can be incorporated in one or more copies, depending on the needs and type of the present case.

Stilbene synthase, which is formed in plants or plant cells with the cooperation of the stilbene synthase gene (or the gene unit) according to the invention, means any enzyme which brings about the formation of such plant antibodies against pests (phytoalexins) which have the stilbene framework.

Preferred stilbenes are pinosylvin (3,5-dihydroxy-stilbene), pterostilbene (3,5-dimethoxy-4'-hydroxystilbene) and resveratrol (3,3 ', 5-trihydroxy-stilbene), with pinosylvin and resveratrol being particularly preferred and Resveratrol is very particularly preferred.

As already mentioned, stilbenes are found in certain tree species as well as in a number of other, preferably dicotyledonous (dicotyledonous) plants. The stilbene synthase gene according to the invention which is preferred is the stilbene synthase gene, which can be isolated from gymnosperms, preferably Pinus, from angiosperms, preferably dicotyledonous (dicotyledonous) plants, in particular from peanut (Arachis hypogaea) and wine (Vitis) and very particularly preferably from peanut .

Particularly preferred as the stilbene synthase gene according to the invention is the stilbene synthase gene, which is present as a gene unit in the plasmid pGS 828.1 (which is described in more detail below), and the essentially identical DNA sequences.

The stilbene synthase gene consists of the 5'- and 3'- untranslated regions and a coding region and is located on a DNA fragment of (ca) 6.7 kbp (gene unit). The gene unit has (3) EcoRI, (3) HindIII and (1) PstI cleavage sites. It can be obtained from the plasmid pGS 828.1 by partial cleavage with EcoRI and HindIII.

The 5′-regulatory part lies next to the first 7 codons of the protein coding region on an approximately 3.3 kbp EcoRI fragment and precedes the rest of the coding region. This region consists of 1540 bp and contains an intron of 369bp and a HindIII interface. The downstream 3'-untranslated region is completely present and is by an EcoRI interface (see Fig. 1) limited. The gene unit in which vector pSP 65 cloned (plasmid pGS 821.1) contains two internal EcoRIs, one PstI and two HindIII cleavage sites and is in the polylinker of the pSP65 plasmid through the SstI cleavage site (directly next to the EcoRI site towards the stilbene synthase gene) delimited at the 5'-end and by the HindIII interface at the 3'-end. The gene unit can be removed from pGS 828.1 particularly advantageously with the aid of SstI and PvuII (FIG. 2), since both interfaces occur only once. The PvuII interface lies outside the actual gene unit. However, this has no influence on the expression of the gene in transgenic plants and the section up to the HindIII site can, if desired, be removed using the customary methods.

The Escherichia coli strain Nurdug 2010, which contains the plasmid pGS 828.1, was obtained from the German Collection of Microorganisms (DSM), Mascheroder Weg 1b, D-3300 Braunschweig, Federal Republic of Germany in accordance with the provisions of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent proceedings and has the deposit number DSM 4243 (filing date: September 17, 1987).

This strain as well as its mutants and variants are also part of the present invention. The plasmid pGS 828.1 deposited in this host can in the usual Ways can easily be obtained in the required amounts by multiplying the strain.

According to the invention, the stilbene synthase gene or the gene unit which contains the DNA sequence coding (below) (proposed) (proposed) ("DNA sequence" (protein-coding region and intron) of the stilbene synthase gene unit from pGS 828.1 "), which is also listed below, is particularly preferred or without the intron or contains essentially equivalent sequences of this DNA sequence It is also part of the present invention which contains essentially equivalent DNA sequences, part of the present invention.

Sequences having essentially the same effect means that DNA or DNA sequences are replaced by other DNA or DNA sequences at one or more locations, but these do not significantly change the result.

Functionally complete genes, such as the stilbene synthase gene according to the invention, consist of a regulatory part (in particular promoter) and the structural gene which codes for the stilbene synthase protein.

Both gene parts can be used independently. It is thus possible to add a different DNA sequence (which differs from the stilbene synthase gene) to the regulatory part, which is to be expressed after incorporation into the plant genome. Since only a few isolated promoters are known which can develop their action in plants or plant cells, the promoter of the stilbene synthase gene which is part of the present invention is a valuable aid in the production of transformed plants or plant cells With the help of the SstI interface and the EcoRI interface at the beginning of the coding region isolated and connected to another gene using the usual methods.

It is also possible to precede the stilbene synthase structure gene with a "foreign" regulatory part. This could be advantageous if only certain (eg plant-specific) regulatory genes can be sufficiently effective in certain plants. The stilbene synthase structural gene (preferably the DNA sequence coding below for stilbene synthase, with or without the intron sequences, and their essentially equivalent sequences) thus represents a valuable, independently usable unit and, as already explained, is likewise Part of the present invention. The stilbene synthase gene according to the invention can be separated into the regulatory part and the structural gene by the usual methods. The complete stilbene synthase gene according to the invention or the gene unit is preferably used.

With the help of the usual methods, it is possible to convert the stilbene synthase gene or the gene unit or its parts one or more times (eg tandem arrangement), preferably simply, into any prokaryotic (preferably bacterial) or eukaryotic (preferably vegetable) DNA as "foreign" "or" additional "DNA. The "modified" DNA, which e.g. can be used for the transformation of plants or plant cells and is contained in plants or plant cells after the transformation, is part of the present invention.

The stilbene synthase gene or the gene unit and / or its parts and the modified DNA can be used as “foreign” or “additional” DNA in vectors (in particular plasmids, cosmids or phages), in transformed microorganisms (preferably bacteria, in particular Gram). negative bacteria, such as E. coli) as well as in transformed plant cells and plants or in their DNA. Such vectors, transformed microorganisms (which may also contain these vectors) and the transformed plant cells and plants and their DNA are constituents of the present invention.

Pests against which resistance or increased resistance can be achieved with the aid of the stilbene synthase gene according to the invention include animal pests such as insects, mites and nematodes and microbial pests such as phytopathogenic fungi and bacteria. Microbial pests, especially phytopathogenic fungi, are particularly emphasized.

The harmful insects include in particular insects of the orders:
Orthoptera, Dermaptera, Isoptera, Thysanoptera, Heteroptera, Homoptera, Lepidoptera, Coleoptera, Hymenoptera and Diptera.

The harmful mites include in particular:
Tarsonemus spp., Panonychus spp. and Tetranychus spp.

The harmful nematodes include in particular:
Pratylenchus spp., Heterodera spp. and Meloidogyne spp.

The microbial pests include in particular the phytopathogenic fungi:
Plasmodiophoromycetes, Oomycetes, Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes, Deuteromycetes.

The phytopathogenic bacteria include in particular the Pseudomonadaceae, Rhizobiaceae, Enterobacteriaceae, Corynebacteriaceae and Streptomycetaceae.

Some pathogens of fungal and bacterial diseases that fall under the generic names listed above may be mentioned as examples, but not by way of limitation:
Xanthomonas species, such as, for example, Xanthomonas campestris pv. Oryzae;
Pseudomonas species, such as, for example, Pseudomonas syringae pv. Lachrymans;
Erwinia species, such as, for example, Erwinia amylovora;
Pythium species, such as, for example, Pythium ultimum;
Phytophthora species, such as, for example, Phytophthora infestans;
Pseudoperonospora species, such as, for example, Pseudoperonospora humuli or Pseudoperonospora cubense;
Plasmopara species, such as, for example, Plasmopara viticola;
Peronospora species, such as, for example, Peronospora pisi or P. brassicae;
Erysiphe species, such as, for example, Erysiphe graminis;
Sphaerotheca species, such as, for example, Sphaerotheca fuliginea;
Podosphaera species, such as, for example, Podosphaera leucotricha;
Venturia species, such as, for example, Venturia inaequalis;
Pyrenophora species, such as, for example, Pyrenophora teres or P. graminea
(Conidial form: Drechslera, Syn: Helminthosporium);
Cochliobolus species, such as, for example, Cochliobolus sativus
(Conidial form: Drechslera, Syn: Helminthosporium);
Uromyces species, such as, for example, Uromyces appendiculatus;
Puccinia species, such as, for example, Puccinia recondita;
Tilletia species, such as, for example, Tilletia caries;
Ustilago species, such as, for example, Ustilago nuda or Ustilago avenae;
Pellicularia species, such as, for example, Pellicularia sasakii;
Pyricularia species, such as, for example, Pyricularia oryzae;
Fusarium species, such as, for example, Fusarium culmorum;
Botrytis species, such as, for example, Botrytis cinerea;
Septoria species, such as, for example, Septoria nodorum;
Leptosphaeria species, such as, for example, Leptosphaeria nodorum;
Cercospora species, such as, for example, Cercospora canescens;
Alternaria species, such as, for example, Alternaria brassicae;
Pseudocercosporella species, such as, for example, Pseudocerco sporella herpotrichoides. Helminthosporium carbonum is also listed.

Plants to which resistance or increased resistance to the above pests can be conferred by incorporating (transforming) the stilbene synthase gene or the gene unit according to the invention include virtually all plants. Naturally, there is a particular need for producing resistance in crop plants such as forest plants, for example spruce, fir, Douglas fir, pine, larch, beech and oak, and plants that supply food and raw materials, for example cereals (in particular wheat, rye, barley, oats, millet, rice) and corn), potatoes, leguminosa such as legumes and in particular alfalfa, soybeans, vegetables (in particular cabbages and tomatoes), fruit (in particular apples, pears, cherries, grapes, citrus, pineapples and bananas), oil palms, tea, cocoa and coffee bushes , Tobacco, sisal and cotton as well as medicinal plants such as Rauwolfia and Digitalis. Potatoes, tomatoes, wine and legumes are particularly preferred.

As already indicated, according to the invention the stilbene synthase gene or the gene unit is built into the natural plant genome one or more times (at the same or different locations in the genome). In plants which already have the ability to synthesize stilbene, the incorporation of one or more stilbene synthase genes according to the invention can lead to significantly improved resistance behavior. If necessary, only the structural gene according to the invention is used, with a regulatory gene possibly isolated from the respective plant being connected upstream.

The increased resistance of the transformed plant cells and plants according to the invention is important for agriculture and forestry, for growing ornamental plants, for growing medicinal plants and for growing plants. Also in the cultivation of plant cells, e.g. for the production of pharmaceutically usable substances, it is advantageous to have plant cells available which have increased resistance to attack by microbial pests, in particular fungi.

• (a) ein oder mehrere Stilbensynthase-Gene bzw. Gen-Einheiten und/oder Teile des Stilbensynthase-Gens bzw. der Gen-Einheit und/oder erfindungsgemäß modifizierte DNA in den Genom von Pflanzenzellen (einschließlich Protoplasten) einsetzt und gegebenenfalls
• (b) aus den transformierten Pflanzenzellen (einschließlich Protoplasten) vollständige transformierte Pflanzen regeneriert und gegebenenfalls
• (c) von den so erhaltenen transformierten Pflanzen die gewünschten Pflanzenteile (einschließlich Samen) gewinnt.

The present invention thus also relates to a process for the production of transformed plant cells (including protoplasts) and plants (including plant parts and seeds), which is characterized in that:

• (a) one or more stilbene synthase genes or gene units and / or parts of the stilbene synthase gene or gene unit and / or DNA modified according to the invention are used in the genome of plant cells (including protoplasts) and, if appropriate
• (b) regenerated complete transformed plants from the transformed plant cells (including protoplasts) and, if appropriate
• (c) the desired plant parts (including seeds) are obtained from the transformed plants thus obtained.

Process steps (a), (b) and (c) can be carried out in a conventional manner by known processes and methods.

Transformed plant cells (including protoplasts) and plants (including plant parts and seeds) which contain one or more stilbene synthase genes or gene units and / or parts of the stilbene synthase gene or gene units as "foreign" or "additional" DNA contain and such transformed plant cells and plants, which are obtainable by the above methods, also belong to the present invention.

• (a) Verwendung des Stilbensynthase-Gens bzw. der Gen-Einheit und/oder seiner Teile und/oder der erfindungsgemäß modifizierten DNA und/oder der erfindungsgemäßen Vektoren und/oder der erfindungsgemäßen transformierten Mikroorganismen zur Transformation von Pflanzenzellen (einschließlich Protoplasten) und Pflanzen (einschließlich Pflanzenteilen und Samen) sowie die
• (b) Verwendung der erfindungsgemäßen transformierten Pflanzenzellen (einschließlich Protoplasten) und Pflanzen (einschließlich Pflanzenteilen und Samen) zur Erzeugung von Vermehrungsmaterial sowie zur Erzeugung neuer Pflanzen und deren Vermehrungsmaterial und allgemein die
• (c) Verwendung des erfindungsgemäßen Stilbensynthase-Gens bzw. der Gen-Einheit und/oder seiner Teile und/oder der erfindungsgemäßen modifizierten DNA zur Bekämpfung von Schädlingen.

Parts of the present invention are also:

• (a) Use of the stilbene synthase gene or the gene unit and / or its parts and / or the DNA modified according to the invention and / or the vectors according to the invention and / or the transformed microorganisms according to the invention for the transformation of plant cells (including protoplasts) and plants (including plant parts and seeds) and the
• (b) Use of the transformed plant cells (including protoplasts) and plants (including plant parts and seeds) according to the invention for the production of propagation material and for the production of new plants and their propagation material and generally the
• (c) Use of the stilbene synthase gene according to the invention or the gene unit and / or its parts and / or the modified DNA according to the invention for controlling pests.

A number of different methods are available for inserting the stilbene synthase gene or the gene unit or its parts as “foreign” or “additional” DNA into the genetic material of plants or plant cells. The gene transfer can be carried out according to the generally known methods, the person skilled in the art being able to determine the appropriate method in each case without difficulty.

The Ti plasmid from Agrobacterium tumefaciens is available as a particularly inexpensive and widely applicable vector for the transfer of foreign DNA into genomes of dicotyledonous and monocotyledonous plants. The genetic material that codes for stilbene synthase is inserted into the T-DNA of suitable Ti pasmids (e.g. Zambryski et al. 1983) and by infection of the plant, infection of plant parts or plant tissues, such as, for example, leaf disks, stems, hypocotyls, cotyledons, meristems and tissues derived therefrom, such as, for example, secondary embryos and calli, or by coculturing protoplasts with Agrobacterium tumefaciens.

An alternative is the incubation of purified DNA containing the desired gene and plant protoplasts (e.g. Davey et al. 1980; Hain et al., 1985; Krens et al., 1982; Paszkowski et al., 1984) in the presence of polycations or Calcium salts and polyethylene glycol.

The DNA uptake can also be promoted by an electric field (electroporation) (e.g. Formm et al. 1986).

The plants are regenerated in a known manner using suitable nutrient media (e.g. Nagy and Maliga 1976).

In a preferred embodiment of the method according to the invention (according to the method from EP-A 116 718) the DNA unit consisting of (approx.) 6.7 kb is made from pGS 828.1, characterized by the corresponding internal EcoRI, HindIII or PstI interfaces (see 1) in a suitable intermediate E. coli vector, for example pGV700, pGV710, (see EP-A-116 718; Deblaere et al. 1986) or preferably derivatives thereof, which additionally contain a reporter gene such as nptII (Herrera-Estrella et al. 1983) or hpt (Van den Elzen et al 1986). The DNA section extracted from pGS 828.1 using SstI and PvuII can also be used for this purpose.

The Escherichia coli strain AZ 4, which contains the vector pGV 710 in an easily isolable form, was obtained from the German Collection of Microorganisms (DSM), Grisebachstrasse 8, D-3400 Göttingen, Federal Republic of Germany in accordance with the provisions of the Budapest Treaty on international use Acknowledgment of the deposit of microorganisms for the purposes of patent proceedings and has the deposit number DSM 3164.

The plasmid so constructed is grown on Agrobacterium tumefaciens, e.g. contains pGV 3850 or derivatives thereof (Zambryski et al. 1983) by conventional methods (e.g. Van Haute et al. 1983). Alternatively, the stilbene synthase gene unit can be cloned in a binary vector (e.g. Koncz and Schell 1986) and transferred to a suitable Agrobacterium strain (Koncz and Schell 1986) as described above. The resulting Agrobacterium strain, which contains the stilbene synthase gene unit in a form which can be transferred to plants, is subsequently used for plant transformation.

In a further preferred embodiment, the isolated plasmid pGS 828.1 is optionally together with another plasmid which contains a reporter gene for plant cells, for example for kanamycin resistance (for example Herrera-Estrella et al. 1983) or a hydromycin resistance (van den Elzen, 1986), preferably pLGV neo 2103 (Hain et al. 1985) , pLGV 23 neo (Herrera-Estrella 1983), pMON 129 (Fraley RT et al., Proc. National Acad. Sci. USA 80 , 4803 (1983), pAK 1003, pAK 2004 (Velten J. et al., EMBO Journ 3, 2723 (1984) or pGSST neo 3 (pGSST3) (EP-A-189 707), in the usual way by direct gene transfer to plant protoplasts (eg Hain et al 1985) circular, but preferably in linear form, when using a plasmid with reporter gene, kanamycin-resistant protoplasts are then checked for expression of stilbene synthase, otherwise (without reporter gene) the resulting calli are checked for expression of the stilbene synthase gene (screening with usual methods).

Transformed (transgenic) plants or plant cells are processed according to known methods, e.g. by leaf disc transformation (e.g. Horsch et al. 1985) by coculturing regenerative plant protoplasts or cell cultures with Agrobacterium tumefaciens (e.g. Marton et al. 1979, Hain et al. 1985) or generated by direct DNA transfection. Resulting transformed plants are either selected by expression for the reporter gene, for example by phosphorylation of kanamycin sulfate in vitro ((Reiss et al. 1984; Schreier et al. 1985) or by expression of nopaline synthase (according to Aerts et al. 1983 ) or stilbene synthase by Northern blot analysis and Western blot analysis proven. The stilbene synthase and the stilbene can also be detected in a known manner with the aid of specific antibodies in transformed plants. Stilbene synthase can also be detected by enzyme activity test (Rolfs et al., Plant Cell Reports 1 , 83-85, 1981).

The cultivation of the transformed plant cells and the regeneration to complete plants is carried out according to the generally customary methods with the aid of the appropriate nutrient media.

Both the transformed plant cells and the transformed plants which contain the stilbene synthase gene or the gene unit according to the invention and which are components of the present invention show a considerably higher resistance to pests, in particular phytopathogenic fungi.

In the context of the present invention, the term "plants" means both whole plants and parts of plants, such as leaves, seeds, tubers, cuttings, etc. "Plant cells" include protoplasts, cell lines, plant calli, etc. "Propagation material" means plants and plant cells which can be used for propagation in transformed plants and plant cells and is therefore also part of the present invention.

In the present context, the expression "essentially equivalent DNA sequences" means that the invention also encompasses those modifications in which the function of the stilbene synthase gene and its parts is not impaired in such a way that stilbene synthase is no longer formed or the regulatory gene part is not becomes more effective. Corresponding modifications can be made by replacing, adding and / or removing DNA segments, individual codons and / or individual nucleic acids.

In the case of the microorganisms which can be used according to the invention, "mutants" and "variants" mean those modified microorganisms which still have the features which are essential for carrying out the invention, in particular contain the respective plasmids.

The present invention will be explained in more detail using the following exemplary embodiments:

1. Isolation of the stilbene synthase gene

Cell cultures from nut plants (Arachis hypogaea) contain the gene for stilbene synthase, which in particular causes the formation of resveratrol synthase (protein size 43,000 D; reaction with specific antiserum).

The peanut cell cultures, the regulation and properties of stilbene synthase, the antiserum against the protein, and the measurement of the enzyme activity are described (Rolfs, CH, Fritzemeier, KH, Kindl, H., Plant Cell Reports 1 , 83-85, 1981; Fritzemeier, KH Rolfs, CH, Pfau, J., Kindl, H., Planta 159 , 25-29, 1983; Schöppner, A., Kindl, H., J. Biol. Chem. 259 , 6806-6811, 1984; see also the abstract: Kindl, H., in: biosynthesis and Biodegradation of Wood Components, Ed.Higuchi, T., Academic Press, Inc., pp. 349-377, 1985).

The following scheme summarizes the expression of the gene and the isolation of the gene for stilbene synthase. The known methods and methods of molecular biology were used for the implementation, as described in detail, for example, in the following manual: Maniatis, T., Fritsch, EF, Sambrook, J .: Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, 1982.

A. Isolation and identification of specific cDNA clones for stilbene synthase.

• (i) die spezifische cDNA hybridisiert mit Stilbensynthase-mRNA,
• (ii) durch Hybridisierung mit der cDNA wird eine mRNA isoliert, die bei in vitro-Übersetzung ein Protein ergibt, welches identisch ist mit Stilbensynthase (Proteingröße 43000 D; Reaktion des Proteins mit Antiserum, welches spezifisch mit Stilbensynthase-Protein reagiert).

First, poly (A) -containing RNA is isolated from peanut cell cultures which synthesize stilbene synthase. The mRNA is then transcribed into DNA using reverse transcriptase and E. coli DNA polymerase; the DNA is cloned into plasmid vector pINIIA with the aid of linkers and propagated in E. coli strain JA221 (Nakamura, K., Inouye, M., EMBO J. 1 , 771-775, 1982). This process results in a "cDNA library": it represents, cloned as DNA in E. coli plasmids, the mRNA population of the peanut cells. It contains the nucleic acid that codes for stilbene synthase, and this is identified by the following steps:

• (i) the specific cDNA hybridizes with stilbene synthase mRNA,
• (ii) hybridization with the cDNA isolates an mRNA which, when translated in vitro, yields a protein which is identical to stilbene synthase (protein size 43000 D; reaction of the protein with antiserum which reacts specifically with stilbene synthase protein).

At the cDNA level, these criteria clearly define the nucleic acid which codes for the stilbene synthase protein.

B. Isolation of the stilbene synthase gene.

First, a "gene library" for nut cells is created: Genomic DNA from enriched cell nuclei (Bedbrook, J., Plant Molecular Biology Newsletter 2 , 24, 1981) is cut with the restriction enzyme SauIIIa so that DNA fragments with a Average length of 10,000-25,000 nucleotide pairs arise. These fragments are cloned into the BamHI site of lambda phage EMBL3 (Frischauf et al., J. Mol. Biol. 170 , 827-842, 1983) and the phages are grown in E. coli. The whole of the phage population contains, cloned in sections, the entire genomic DNA of the peanut cells, and thus also the gene for stilbene synthase.

The gene for stilbene synthase, its mRNA and the stilbene synthase cDNA contain the same nucleic acid sequences since they are derived from one another (gene → mRNA → cDNA). This means that the gene for stilbene synthase can be identified by specific hybridization with stilbene synthase cDNA or mRNA. The phages with the gene are identified by hydridation, then isolated and propagated. The genomic DNA from peanut cells cloned in this phage is further analyzed by analysis with various Restriction enzymes are mapped and the position of the stilbene synthase gene is determined by further hybridization experiments with the cDNA. Finally, the gene unit is cut out of the phage by partial digestion with EcoRI and HindIII, cloned in the correspondingly cut plasmid vector pSP65 (from Amersham Buchler GmbH & Co. KG, Braunschweig, Federal Republic of Germany), and propagated as a recombinant plasmid. This plasmid is referred to as pGS 828.1.

2. Description of the plasmid pGS 828.1 (see FIGS. 1-2)

• (i) Gen-Einheit Stilbensynthase: Das gesamte Gen, bestehend aus Struktur-Gen und Regulator-Anteil (beide aus Erdnußzellen) befindet sich auf einer Nucleinsäure, die als DNA-Fragment von (ca) 6700 Nukleotidpaaren durch partielle Spaltung mit den Restriktionsenzymen EcoRI und HindIII aus dem Plasmid herausgeschnitten wird. Die Gen-Einheit enthält Schnittstellen für die Restriktionsenzyme EcoRI, HindIII, und PstI.
• (ii) Vektor-Plasmid: Die Gen-Einheit ist in Vektor pSP65 kloniert. Die Größe des Vektors ist 3000 Nukleotidpaare. Er trägt das Gen für Ampicillin-Resistenz, d.h. E. coli-Zellen mit diesem Plasmid wachsen in Nährmedien, die das Antibiotikum Ampicillin enthalten. Ori: Bezeichnung für Sequenzen, die für die Vermehrung des Plasmids in E. coli notwendig sind.

The plasmid consists of two components:

• (i) Gene unit stilbene synthase: The entire gene, consisting of the structural gene and regulator part (both from peanut cells) is located on a nucleic acid, which is a DNA fragment of (approx) 6700 nucleotide pairs by partial cleavage with the restriction enzymes EcoRI and HindIII is excised from the plasmid. The gene unit contains interfaces for the restriction enzymes EcoRI, HindIII, and PstI.
• (ii) Vector plasmid: The gene unit is cloned into vector pSP65. The size of the vector is 3000 nucleotide pairs. It carries the gene for ampicillin resistance, ie E. coli cells with this plasmid grow in Culture media containing the antibiotic ampicillin. Ori: Name for sequences which are necessary for the multiplication of the plasmid in E. coli.

The plasmid carries a gene for ampicillin resistance and contains 9350 nucleotide pairs (9.35 kBp). In E. coli cells which contain pGS 828.1 (E. coli Nurdug 2010), they can be propagated in the usual way.

Preferred nutrient medium for E. coli cells (e.g. JA221, Nakamura, K., Inouye, M., EMBO J. 1 , 771-775, 1982) which contain pGS 828.1 (E. coli Nurdug 2010): Bacto peptone * 10 g Yeast extract 5 g NaCl 5 g Agar 20 g H₂O 1 l pH 7.5 Fermentation: 37 ° C, aerobic (* Bacto is a trademark of DIFCO Lab. Detroit, USA).

3. Tobacco transformation a) Culture of tobacco sprouts and isolation of tobacco protoplasts:

Nicotiana tabacum (Petit Havanna SR1) is propagated as a sterile sprout culture on hormone-free LS medium (Linsmaier and Skoog 1965). At intervals of approx. 6-8 weeks, shoot sections are transferred to fresh LS medium. The shoot cultures are kept in a culture room at 24-26 ° C under 12 h light (1000-3000 lux).

For the isolation of leaf protoplasts, approx. 2 g of leaves (approx. 3-5 cm long) are cut into small pieces (0.5 cm x 1 cm) with a fresh razor blade. The leaf material is in 20 ml enzyme solution, consisting of K3 medium (Nagy and Maliga 1976), 0.4 m sucrose, pH 5.6, 2% cellulase R10 (Serva), 0.5% Macerozym R10 (Serva) for 14- Incubated for 16 h at room temperature. The protoplasts are then separated from cell residues by filtration through 0.30 mm and 0.1 mm steel sieves. The filtrate is centrifuged at 100 xg for 10 minutes. During this centrifugation, intact protoplasts float and collect in a band at the top of the enzyme solution. The pellet from cell residues and the enzyme solution are aspirated with a glass capillary. The pre-cleaned protoplasts are made up to 10 ml with fresh K3 medium (0.4 M sucrose as an osmotic agent) and floated again. The washing medium is suctioned off and the protoplasts are diluted to 1-2 x 10⁵ / ml for culture or subsequent infection with agrobacteria (coculture). The protoplast concentration is determined in a counting chamber.

b) Transformation of regenerating tobacco protoplasts by coculture with Agrobacterium tumefaciens:

The method of Marton et al. 1979 used with minor changes. The protoplasts are isolated as described and in a density of 1-2 x 10⁵ / ml in K3 medium (0.4 m sucrose, 0.1 mg / l NAA, 0.2 ml in K3 medium (0.4 m sucrose, 0.1 mg / l NAA, 0.2 mg kinetin) incubated for 2 days in the dark and for one to two days under low light (500 lux) at 26 ° C. As soon as the first division of the protoplasts occurs, 30 µl of an agrobacterial suspension are added minimally A (Am) medium (density approx. 10⁹ agrobacteria / ml) to 3 ml regenerating protoplasts. The coculture time is 3-4 days at 20 ° C in the dark. Then the tobacco cells are filled in 12 ml centrifuge tubes, with sea water (600 mOsm / kg) diluted to 10 ml and pelleted at 60 xg for 10 minutes, this washing process is repeated 1-2 more times to remove most of the agrobacteria. The cell suspension is in a density of 5 x 10⁴ / ml in K3 medium (0.3 M sucrose) with 1 mg / l NAA (naphthyl-1-acetic acid), 0.2 ml / l kinetin and 500 mg / l of cephalospo rin antibiotic cefotaxime cultivated. The cell suspension is diluted with fresh K3 medium every week and the osmotic value of the medium gradually reduced by 0.05 m sucrose (approx. 60 mOsm / kg) per week. The selection with kanamycin (100 mg / l kanamycin sulfate (Sigma), 660 mg / g active km) is started 2-3 weeks after the coculture in agarose bead type culture (Shillito et al. 1983). Colonies resistant to kanamycin can be distinguished from the background of remaining colonies 3-4 weeks after the start of the selection.

c) Direct transformation of tobacco protoplasts with DNA. Calcium nitrate-PEG transformation.

Approximately 10⁶ protoplasts in 180 µl K3 medium with 20 µl aqueous DNA solution containing 0.5 µg / µl pGS 828.1 and 0.5 µg / µl pLGV neo 2103 (Hain et al. 1985) are carefully mixed in a Petri dish. Then 200 μl of fusion solution (0.1 m calcium nitrate, 0.45 M mannitol, 25% polyethylene glycol (PEG 6000), pH 9) are carefully added. After 15 minutes, 5 ml of washing solution (0.275 M calcium nitrate pH 6) are added and after a further 5 minutes the protoplasts are transferred into a centrifuge tube and pelleted at 60 x g. The pellet is taken up in a small amount of K3 medium and cultivated as described in the next section. Alternatively, the protoplasts according to Hain et al. 1985 transformed.

The transformation can also be carried out without the addition of 0.5 µg / µl pLGV neo 2103. Since no reporter gene is used in this case, the resulting calli were checked for the presence of the stilbene synthase gene unit using dot-blot hybridization. An internal EcoRI-HindIII fragment from pGS 828.1 can be used as a hybridization sample. Of course, other detection methods, such as testing with antibodies or determining fungicide resistance, can also be used.

d) Culture of protoplasts incubated with DNA and selection of kanamycin-resistant calli:

A modified "bead type culture" technique (Shillito et al. 1983) is used for the culture and selection of kanamycin-resistant colonies described below. One week after treatment of the protoplasts with DNA (see c), 1 ml of the cell suspension with 3 ml of K3 medium (0.3 M sucrose + hormones; 1.2% (Seaplaque) LMT agarose (low melting agarose, marine colloids) in 5 cm petri dishes mixed. For this purpose, agarose is dry autoclaved and briefly boiled in the microwave after adding K3 medium. After the agarose has solidified, the agarose slices ("beads") with the embedded tobacco microcalli are transferred to 10 cm petri dishes for further culture and selection Add 10 ml of K3 medium (0.3 M sucrose, 1 mg / l NAA, 0.2 mg / l kinetin) and 100 mg / l kanamycin sulfate (Sigma). The liquid medium is changed every week, whereby the osmotic value of the Medium gradually reduced.

The exchange medium (K3 + Km) is reduced by 0.05 m of sucrose (approx. 60 mOsm) per week.

e) Regeneration of kanamycin-resistant plants:

As soon as the kanamycin-resistant colonies have reached a diameter of approx. 0.5 cm, half is placed on regeneration medium (LS medium, 2% sucrose, 0.5 mg / l benzylaminopurine BAP) and exposed to 12 h light (3000-5000 lux ) and 24 ° C in the culture room. The other half is called Callus culture propagated on LS medium with 1 mg / l NAA, 0.2 mg / l kinetin, 0.1 mg / l BAP and 100 mg / l kanamycin sulfate. When the regenerated shoots are approx. 1 cm in size, they are cut off and placed on 1/2 LS medium (1% sucrose, 0.8% agar) without growth regulators for rooting. The shoots are rooted on 1/2 MS medium with 100 mg / l kanamycin sulfate and later converted into soil.

f) transformation of leaf disks by Agrobacterium tumefaciens

For the transformation of leaf discs (Horsch et al. 1985) leaves of approx. 2-3 cm in length are punched out of sterile shoot cultures into discs of 1 cm in diameter and with a suspension of appropriate Agrobacteria (approx. 10⁹ / ml) (cf. b) in Incubate on medium, see below) for approx. 5 minutes. The infected leaf pieces are kept on MS medium (see below) without hormones at 3-4 ° C for 3-4 days. During this time, Agrobacterium grows over the leaf pieces. The leaf pieces are then washed in MS medium (0.5 mg / ml BAP, 0.1 mg / ml NAA) and on the same medium (0.8% agar) with 500 μg / ml cefotaxime and 100 μg / ml kanamycin sulfate (Sigma) placed. The medium should be replaced after two weeks. Transformed shoots become visible after another 2-3 weeks. The regeneration of shoots should also be carried out in parallel without selection pressure. The regenerated shoots must then go through biological Tests eg for nopaline synthase or stilbene synthase activity can be tested for transformation. In this way 1-10% transformed shoots are obtained.

Biochemical detection method of transformation Detection of nopaline in plant tissues:

As with Otten and Schilperoort (1978) and Aerts et al. (1979) as follows. 50 mg of plant material (callus or leaf pieces) are incubated overnight in LS medium with 0.1 M arginine at room temperature in an Eppendorf tube. The plant material is then dabbed onto absorbent paper, homogenized in a fresh Eppendorf centrifuge tube with a glass rod and centrifuged for 2 minutes in an Eppendorf centrifuge. 2 .mu.l of the supernatant are spotted on a paper suitable for electrophoresis (Whatman 3 MM paper) (20 x 40 cm) and dried. The paper is soaked with the eluent (5% formic acid, 15% acetic acid, 80% H₂O, pH 1.8) and electrophoresed at 400 V for 45 minutes. Nopalin runs towards the cathode. The paper is then hot dried with an air stream and drawn in the direction of travel through phenanthrenequinone colorant (equal volume 0.02% phenanthrenequinone in ethanol and 10% NaOH in 60% ethanol). The dried paper is viewed and photographed under long-wave UV light. Arginine and arginine derivatives are stained yellow fluorescent with the reagent.

Neomycin phosphotransferase (NPT II) enzyme test:

NPT II activity in plant tissue is determined by in situ phosphorylation of kanamycin, as described by Reiss et al. (1984) and by Schreier et al. (1985) modified as follows. 50 mg of plant tissue are homogenized in 50 .mu.l extraction buffer (10% glycerol, 5% 2-mercaptoethanol, 0.1% SDS, 0.025% bromophenol blue, 62.5 mM Tris pH 6.8) with the addition of glass powder on ice and for 10 minutes centrifuged in an Eppendorf centrifuge at 4 ° C. 50 µl of the supernatant are placed on a native polyacrylamide gel (145 x 110 x 1.2 mm; separating gel: 10% acrylamide, 0.33% bisacrylamide, 0.375 M Tris pH 8.8, bulk gel: 5% acrylamide, 0.165% bisacrylamide, 0.125 M Tris pH 6.8) and electrophoresed overnight at 4 ° C and 60 V. As soon as the bromophenol blue marker runs out of the gel, the gel is washed twice with distilled water for 10 minutes and once for 30 minutes with reaction buffer (67 mM Tris-maleate, pH 7.1, 42 mM MgCl₂, 400 mM ammonium chloride). The gel is placed on a glass plate of the same size and covered with 40 ml of 1% agarose in reaction buffer which contains the substrates kanamycin sulfate (20 µg / ml) and 20-200 µCi ³²P ATP (Amersham). The sandwich gel is incubated for 30 minutes at room temperature and then a sheet of phosphocellulose paper P81 (Whatman) is placed on the agarose. Over it are four layers of filter paper 3 MM, (Whatman) and some paper towels stacked. The transfer of in situ phosphorylated radioactive kanamycin phosphate to the P81 paper is stopped after 3-4 hours. The P81 paper is for 30 min. incubated in a solution of Proteinase K and 1% sodium dodecyl sulfate (SDS) at 60 ° C and then washed 3-4 times in 250 ml of 10 mM phosphate buffer pH 7.5 at 80 ° C, dried and for 1-12 h at -70 ° C autoradiographed (XAR5 film Kodak).

4. Transformation of Medicago Sativa (alfalfa) a) Plant material

The Medicago sativa plant (Regen S, clone RA₃ Walker et al, 1978) was cultivated as a sterile shoot culture on LS medium (Linsmaier and Skoog, 1965), in the long day (16 h light, 8 h dark) at 26 ± 2 ° C.

b) Culture conditions

Glass jars (250 ml - 1.5 l) with loose glass lids served as culture jars for shoot cultures. Plastic petri dishes were used for all other plant cultures (embryo, callus, protoplasts).

The plants and tissue cultures, with the exception of the protoplasts, were cultivated in culture rooms in the long day (16 h light, 8 h dark) at 26 ± 2 ° C. The fluorescent tubes had the light color Universal White (Osram L58W / 25). The distance between the tubes and the cultures was 10-30 cm, which corresponds to 1500-4500 lux light intensity. The air humidity remained unregulated. The protoplasts were cultivated in culture cabinets at a maximum of 500 lux and 26 ° C.

c) Callus culture

• 1 min in 70% Ethanol
• 10 min in 10% handelsüblichem Desinfektionsmittel (z.B. Dan Klorix)
• 3 Waschungen in sterilem Leitungswasser.

Callus was induced by petioles of the greenhouse plants. Approx. 5 cm petioles were cut from the greenhouse plants with a scalpel. The petioles were first surface sterilized:

• 1 min in 70% ethanol
• 10 min in 10% commercial disinfectant (e.g. Dan Klorix)
• 3 washes in sterile tap water.

• 1. B₅h (Atanassov und Brown, 1984)
• 2. SHR: SH (Shenk und Hildebrandt, 1972) mit 25 µM (4,655 mg/l) NAA und 10 µM (2,15 mg/l) Kinetin (Walker und Sato, 1981)
• 3. B₅H₃: B₅ (Gamborg et al., 1968) mit 2,6 µM (0,5 mg/l) NAA, 2,2 µM (0,5 mg/l) BAP, 2,2 µM (0,5 mg/l) 2,4D (Oelck, Dissertation 1984).

After sterilization, the petioles were cut into 1-1.5 cm long pieces and placed in petri dishes on solid agar medium. Three different media were used for callus induction and further culture:

• 1. B₅h (Atanassov and Brown, 1984)
• 2. SHR: SH (Shenk and Hildebrandt, 1972) with 25 µM (4.655 mg / l) NAA and 10 µM (2.15 mg / l) kinetin (Walker and Sato, 1981)
• 3. B₅H₃: B₅ (Gamborg et al., 1968) with 2.6 µM (0.5 mg / l) NAA, 2.2 µM (0.5 mg / l) BAP, 2.2 µM (0.5 mg / l) 2.4D (Oelck, dissertation 1984).

After three weeks, the outer parts of the callus were cut off with a scalpel and subcultured on fresh medium.

d) Callus regeneration

The regeneration of plants from callus was carried out according to the protocol of Stuart and Strickland, 1984 a, b, with modifications.

Somatic embryogenesis was induced by incubation of callus tissue in liquid SH medium (Shenk and Hildebrandt, 1972) containing 50 µM (11 mg / l) 2.4D and 5 µM (1.07 mg / l) kinetin. In an Erlenmeyer flask (100 ml in a 500 ml flask) 30 mg callus (fresh weight) were added per ml medium. The induction was carried out for 3-4 days on a shaker (100 rpm) at 26 ° C in the plant culture room. The callus path was then separated from the medium on a sieve (850 μm).

It was pressed through the sieve with a spatula and small clusters of cells were collected on a sieve underneath with a mesh size of 250 μm 2. The cell clusters per 100 ml of induction medium were washed with 500 ml of SHJ medium without hormones (SH). The washing solution was removed by draining as far as possible (approx. 5 min). The fresh weight was determined and the cell clusters were resuspended in SH medium. 75 mg in 0.5 ml were applied in a pipette to approximately 10 ml of solid regeneration medium SHR. The regeneration medium SHR consisted of SH medium with 25 mM NH₄⁺ and 100 mM L-proline in 3% sucrose.

After approximately four weeks, well developed embryos with a clear polarity (cotyledon stage, Dos Santos et al., 1983) were placed on solid 1 / 2SH medium with 25 µM (8.6 mg / l) gibberilinic acid (GA₃) and 0.25 µM ( 0.046 mg) NAA. After rooting and developing a sprout with leaves, the small plants were transferred to LS medium.

The table shows the composition of the media B₅h, SHJ, SHR, 1/2 SH, LS. The liquid medium SH corresponds to the SHJ medium without the hormones 2,4D and kinetin. The amounts are given in mg / l unless otherwise noted.

The media were sterilized by heating in an autoclave for 17 min at 121 ° C. Kinetin, L-gluthathione and amino acids were sterilized with a filter and added to the medium at 60 ° C. after heating in an autoclave.

e) Protoplast culture

• Zuerst wurden die Blätter in einer Petrischale mit EMI (Atanassov und Brown, 1984) befeuchtet und mit einer neuen Rasierklinge fein zerschnitten.
• 1-1,5 g Blätter wurden dann mit 10 ml Enzymlösung in einer Petrischale (⌀ 10 cm) für 3-4 h inkubiert. Die Inkubation erfolgte bei 26°C und schwacher Beleuchtung. Die Freisetzung von Protoplasten aus den Blättern wurde unter dem Mikroskop verfolgt. Alle 30 min wurde die Schale 2-3 mal geschwenkt.

The leaves served as the starting material for the isolation of protoplasts, sterile shoot cultures 2-3 months old. The harvest took place 2-3 hours after the light was switched on.

• First the leaves were moistened in a petri dish with EMI (Atanassov and Brown, 1984) and finely cut with a new razor blade.
• 1-1.5 g of leaves were then incubated with 10 ml of enzyme solution in a petri dish (⌀ 10 cm) for 3-4 h. The incubation was carried out at 26 ° C and weak lighting. The release of protoplasts from the leaves was followed under the microscope. The bowl was swung 2-3 times every 30 minutes.

• 200 mg Cellulose Onozuka R10
• 80 mg Macerozyme R10      Serva
• 10 mg Pectolyase Y-23      Sigma
• 540 mg Sorbitol

The enzyme solution consisted of a 1: 1 mixture of the protoplast culture medium (AP) from Atanassov and Brown (1984) with the hormones 0.2 mg / l 2.4D, 0.5 mg / l zeatin and 1 mg / l NAA and an enzyme solution. The enzyme solution (Kao and Michayluk, 1979; modified) consisted of:

• 200 mg cellulose Onozuka R10
• 80 mg Macerozyme R10 Serva
• 10 mg pectolyase Y-23 Sigma
• 540 mg sorbitol

f) Transformation of an induced callus

Callus was induced for embryogenesis using the method described under callus regeneration.

Following the 3-4 day incubation in liquid SHJ, the callus material was washed on a sieve (mesh size 100 μm) with liquid SHR, which contained no agar and no L-proline. The callus material was then taken up in liquid SHR. Approx. 1 g of callus material was added to 10 ml of medium.

After the addition of the agrobacteria which contained Ti plasmids carrying the stilbene synthase gene (2x10⁷ / ml final concentration), the callus material was incubated on a shaker (90 rpm) for 2-3 days at 26 ° C.

The material was then washed on a sieve (mesh size 100 μm 2) with liquid SHR.

The plating (75 mg callus / 10 ml medium) was carried out on the normal solid SHR with 100 mM L-proline. In addition to the selective antibiotics, the medium in the plates contained 500 µg / ml Claforan. After four weeks, the resistant structures were placed on fresh medium containing antibiotics and a further three weeks later they were divided and half was placed on fresh medium without selective antibiotics, the other half on medium containing antibiotics.

a) Transformation of embryos

The transformation of embryos was carried out analogously to the transformation of the induced callus. Embryos 4-5 weeks old were used as starting material. They were finely cut in a Petri dish with a razor blade and then washed on a sieve (mesh size 100 µm) with liquid SHR. Approx. 1 g of cut embryos were taken up in 10 ml of liquid SHR. After the addition of agrobacteria (2 × 10⁷ / ml final concentration), the incubation was carried out on a shaker (90 rpm) for 2-3 days at 26 ° C. The embryo pieces were then washed on a sieve (100 μm 2) with liquid SHR. The plating was done with a spatula on plates that the normal solid SHR was using Contained 100 mM L-proline. Approx. 50-100 mg pieces of embryo were distributed per plate containing 10 ml medium. In addition to the selective antibiotics, the medium in the plates contained 500 µg / ml Claforan. Three weeks after plating, the secondary embryos were subcultured on fresh plates. Well-developed embryos were placed on antibiotic-free 1/2 SH medium (Stuart and Strickland, 1984, b) to enable further development into plants. Small plantlets with roots were then transferred to LS medium.

5. Transformation of Solanum tuberosum (potato)

The transformation was transformed exactly in the manner specified in EP-A-0 242 246, pages 14 to 15, the agrobacteria containing Ti plasmids which carry the stilbene synthesis gene.

All percentages in the above examples relate to percentages by weight, unless stated otherwise.

In the plant cells and plants (tobacco) obtained according to the above examples, the presence of the stilbene synthase gene was confirmed by Southern blot analysis. Expression of the stilbene synthase gene was detected by Northern blot analysis, stilbene synthase and stilbene with the help of specific antibodies. Transformed and non-transformed plants (for comparison) were sprayed with a spore suspension of Botrytis cinera and the fungal infection was rated after 1 week. The transformed plants showed (compared to the non-transformed comparison plants) an increased resistance to fungal attack. Corresponding positive results were also found for Medicago sativa and potatoes.

In the following, the DNA sequence (protein coding region and intron) of the stilbene synthase gene with a part of the 5′- and 3′- untranslated regions is listed, as it is present in the plasmid pGS 828.1. The restriction sites for EcoRI, PstI and HindIII are given. The corresponding protein sequence is given in the one-letter code.

Some of the media used in the transformation of plants or plant cells are described below:

Am medium

3.5 g K₂HPO₄ 1.5 g KH₂PO₄ 0.5 g Na₃ citrate 0.1 g MgSO₄ x 7H₂O 1 g (NH₄) ₂SO₄ 2 g Glucose ad 1 l

Medium for sterile shoot culture of tobacco

K3 medium

On the culture of Nicotiana tabacum petit Havana SR1, Nicotiana tabacum Wisconsin 38, and Nicotiana plumaginifolia protoplasts (Nagy and Maliga, 1976) Macro elements NH₄NO₃ 250 mg / l KNO₃ 2500 mg / l CaCl₂ · 2H₂O 900 mg / l MgSO₄.7H₂O 250 mg / l NaH₂PO₄.1H₂O 150 mg / l (NH₄) ₂SO₄ 134 mg / l CaHPO₄.1H₂O 50 mg / l Micro elements H₃BO₃ 3 mg / l MnSO₄.1H₂O 10 mg / l ZnSO₄.4H₂O 2 mg / l AI 0.75 mg / l Na₂MoO₄.2H₂O 0.25 mg / l CuSO₄.5H₂O 0.025 mg / l CoCl₂.6H₂O 0.025 mg / l Fe-EDTA Na₂EDTA 37.2 mg / l FeSO₄.7H₂O 27.8 mg / l Inositol 100 mg / l Sucrose 137 g / l (= 0.4 M) Xylose 250 mg / l Vitamins Nicotinic acid 1 mg / l Pyridoxine 1 mg / l Thiamine 10 mg / l Hormones NAA 1.0 mg / l Kinetin 0.2 mg / l pH 5.6 Sterilize the filter

Linsemaier and Skoog Medium (Linsemaier and Skoog 1965)

• Thiamin wird in höherer Konzentration eingewogen 0,4 mg/l anstatt 0,1 mg/l;
• Glycin, Pyridoxin und Nicotinsäure fehlen.

Macro-elemente NH₄NO₃ 1650 mg/l KNO₃ 1900 mg/l CaCl₂.2H₂O 440 mg/l MgSO₄.7H₂O 370 mg/l KH₂PO₄ 170 mg/l Micro-elemente H₃BO₃ 6,2 mg/l MnSO₄.1H₂O 22,3 mg/l ZnSO₄.4H₂O 8,6 mg/l KI 0,83 mg/l Na₂MoO₄.2H₂O 0,25 mg/l CuSO₄.5H₂O 0,025 mg/l CoCl₂.6H₂O 0,025 mg/l Fe-EDTA Na₂EDTA 37,2 mg/l FeSO₄.7H₂O 27,8 mg/l Inosit Thiamin 100 mg/l Saccharose 30 g/l Agar 8 g/l Vitamine 0,4 mg/l Hormone: NAA 1 mg/l Kinetin 0,2 mg/l pH 5,7 vor dem Autoklavieren For the culture of regenerating protoplasts and for tissue culture of tobacco tumors and callus. Linsemaier and Skoog (LS) Medium is Murashige and Skoog Medium (Murashige and Skoog, 1962) with the following modifications:

• Thiamine is weighed in a higher concentration 0.4 mg / l instead of 0.1 mg / l;
• Glycine, pyridoxine and nicotinic acid are missing.

Macro elements NH₄NO₃ 1650 mg / l KNO₃ 1900 mg / l CaCl₂.2H₂O 440 mg / l MgSO₄.7H₂O 370 mg / l KH₂PO₄ 170 mg / l Micro elements H₃BO₃ 6.2 mg / l MnSO₄.1H₂O 22.3 mg / l ZnSO₄.4H₂O 8.6 mg / l AI 0.83 mg / l Na₂MoO₄.2H₂O 0.25 mg / l CuSO₄.5H₂O 0.025 mg / l CoCl₂.6H₂O 0.025 mg / l Fe-EDTA Na₂EDTA 37.2 mg / l FeSO₄.7H₂O 27.8 mg / l Inositol Thiamine 100 mg / l Sucrose 30 g / l Agar 8 g / l Vitamins 0.4 mg / l Hormones: NAA 1 mg / l Kinetin 0.2 mg / l pH 5.7 before autoclaving

The following literature can be cited for the transformation of plants or plant cells:
Aerts M, Jacobs M, Hernalsteens JP, Van Montagu M, Schell J (1983) Induction and in vitro culture of Arabidopsis thaliana crown gall tumors, Plant Sci Lett. 17: 43-50
Atamasov A, Brown DCW (1984) Plant regeneration from suspension culture and mesophyll protoplasts of Medicago sativa L. Plant Cell Tiss Org. Cult. 3, 149-162
Czernilofsky et al. (1986) Studies of the Structure and Functional Organization of Foreign DNA Integrates into the Genome of Nicotiana tabacum. DNA, Vol. 5, No. 6: 473 (1986)
Davey MR, Cocking EC, Freeman J, Pearce N, Tudor I (1980) Transformation of Petunia protoplasts by isolated Agrobacterium plasmid. Plant Sci Lett 18: 307-313
Deblaere R., Bytebier B., De Greve H, Deboeck F, Schell J, van Montagu M, Leemans J (1985) Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic Acid Research, Vol. 13, No. 13, 4777 (1985)
Fromm ME, Taylor LP, Walbot V (1986) Stable transformation of maize after gene transfer by electroporation. Nature 319: 791-793
Hain, R., Stabel, P., Czernilofsky, A.Pp., Steinbiß, HH, Herrera-Estrella, L., Schell, J. (1985) Uptake, integration, expression and genetic transmission of a selectable chimeric gene by plant protoplasts. Molec Gen Genet 199: 161-168
Hernalsteens JP, Thia-Tong L, Schell J, Van Montagu M (1984) An Agrobacterium-transformed Cell culture from the monocot Asparagus officinalis. EMBO J 3: 3039-3041
Herrera-Estrella L., De Block M., Messens E., Hernalsteens JP., Van Montagu M., Schell J. (1983) EMBO J. 2 : 987-995.

Hooykaas-Van Slogteren GMS, Hooykaas PJJ, Schilperoort RA (1984) Expression of Ti-plasmid genes in monocotyledonous plants infected with Agrobacterium tumefaciens Nature 311: 763-764
Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 277: 1229-1231
Kao KN, Michayluk MR (1980) Plant regeneration from Mesophyll protoplasts of Alfalfa. Z plant physiol. 96, 135-141
Keller WA, Melchers G (1973) The effect of high pH and calcium on tobacco leaf protoplast fusion. Z Naturforschg 28c: 737-741
Krens FH, Molendijk L, Wullems GJ, Schilperoort RA (1982) in vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296: 72-74
Koncz C, Schell J (1986) The promotor of T _L -DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a noval type of Agrobacterium linary vector. Mol. Gen. Genet. (1986) 204: 338-396
Linsmaier DM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18: 100-127
Marton L, Wullems GJ, Molendijk L, Schilperoort PR (1979) In vitro transformation of cultured cells from Nicotiana tabacum by Agrobacterium tumefaciens. Nature 277: 1229-131
Nagy JI, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z. Plant Physiol 78: 453-455
Otten LABM, Schilperoort RA (1978) A rapid microscale method for the detection of Lysopin and Nopalin dehydrogenase activities. Biochim biophys acta 527: 497-500
Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I (1984) Direct gene transfer to plants. EMBO J 3: 2717-2722
Potrykus I, Saul MW, Petruska J, Paszkowski J, Shillito RD (1985) Direct gene transfer to cells of a gramineous monocot. Molec genet 199: 183-188
Shenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous cell cultures. Approx. J. Bot. 50, 199-204
Shillito RD, Paszkowski J. Potrykus I (1983) Agarose plating and Bead type culture technique enable and stimulate development of protoplast-derived colonies in an number of plant species. Pl Cell Rep 2: 244-247
Simons-Schreier A, Dissertation University of Cologne (1988) Studies on the transformation of Medicago sativa and the expression of a leg hemoglobin gene (1bc3) and a chimeric 1b-cat gene during symbiosis in transgenic plants
Stuart DA, Strickland SG (1984) Somatic embryogenesis from cell cultures of Medicago sativa LI The role of amino acid additions to the regeneration medium. Plant Sci. Let. 34, 165-174
Stuart DA, Strickland SG (1984) Somatic embryogenesis from cell cultures of Medicago sativa L. II. The interaction of amino acids with ammonium. Plant Sci. Let. 34, 175-181
Van den Elzen PJM, Townsend J, Lee KY, Bedbrook JR (1985) Achimaeric resistance gen as a selectable marker in plant cells. Plant Mol. Biol. 5, 299-302.

Velten J, Velten L, Hain R, Schell J (1984) Isolation of a dual plant promotor fragment from the Ti Plasmid of Agrobacterium tumefaciens. EMBO J 12: 2723-2730
Walker KA, Yu PC, Sato SJ, Jaworski EG (1978) The hormonal control of organ formation in callus of Medicago sativa L. cultured in vitro. Amer. J. Bot. 65 (6), 654-659
Walker KA, Sato SJ (1981) Morphogenesis in callus tissue of Medicago sativa: The role of ammonium ion in somatic ambryogenesis. Plant Cell Tiss. Org. Cult. 1, 109-121
Wullems GJ, Molendijk L, Ooms G, Schilperoort RA (1981) Differential expression of crown gall tumor markers in transformants obtained after in vitro Agrobacterium tumefaciens - induced transformation of cell wall regenerating protoplasts derived from Nicotiana tabacum. Proc Natl Acad Sci 78: 4344-4348
Zambryski P, Joos H, Genetello C, van Montagu M, Schell J (1983) Ti-plasmid vector for the introduction of DNA into plant cells without altering their normal regeneration capacity, EMBO J 12: 2143-2150.

Bernd Reiss, Rolf Sprengel, Hans Will and Heinz Schaller (1984) A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell tracts, GENE 1081: 211-217
Peter H. Schreier, Elisabeth A. Seftor, Jozef Schell and Hans. J. Bohnert (1985) The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts, EMBO J Vol. 4, No. 1: 25-32
Gamborg OL, Miller RA and Ojima K. (1968) Nutrient requirements of suspension cultures of soybean root cells, Experimental Cell Research 50: 151-158
Michael M. Oelck, dissertation, 1984, University of Cologne, Federal Republic of Germany, regeneration of legumes from tissue and cell cultures
The following published patent applications can also be listed:
EP-A 116 718
EP-A 159 418
EP-A 120 515
EP-A-120 516
EP-A-172 112
EP-A-140 556
EP-A-174 166
EP-A-122 791
EP-A-126 546
EP-A-164 597
EP-A-175 966
WO 84/02913
WO 84/02919
WO 84/02920
WO 83/01176

Explanations to FIGS. 1 and 2

Fig. 1

represents the nucleic acid segment with 6700 nucleotide pairs obtained from the plasmid pGS 828.1 by partial cleavage with EcoRI and HindIII and which contains the stilbene synthase gene.
(1) and (2) indicate the beginning (1) and end (2) of the structural gene. The regulatory part of the gene is on the left section (left of (1)). The following restriction enzyme interfaces are indicated:
E: EcoRI
H: Hind III
P: PstI
S: SstI

Fig. 2

represents the gene unit according to FIG. 1 in the vector pSP 65. The entire plasmid is designated pGS828.1.
"ORI" means Origin of Replication (the sequences in the vector pSP65 which are important for the multiplication of the plasmid in Escherichia coli). "AMP ^R " stands for the gene which brings about the resistance of pSP65-containing Escherichia coli to ampicillin. In addition to the restriction interfaces shown in FIG. 1, the PvuII interface designated as Pv is indicated.

Claims (21)

1. Stilbene synthase gene corresponding to the stilbene synthase gene which is contained in plasmid pGS 828.1 (obtainable from E. coli DSM 4243), and the DNA sequences derived therefrom with stilbene synthase activity.
2. Stilbene synthase gene according to Claim 1, in which stilbene stynthase means resveratrol synthase and pinosylvine synthase.
3. Stilbene synthase gene according to Claims 1 and 2, which can be obtained from groundnut (Arachis hypogaea), Pinus and vine.
4. Stilbene synthase gene unit which consists of approximately 6,700 base pairs and exhibits 3 EcoRI, 3 HindIII and 1 PstI cleavage sites, and which can be obtained by partial cleavage of the plasmid pGS 828.1 (obtainable from E. coli DSM 4243) using EcoRI and HindIII, or from plasmid pGS 828.1 (obtainable from E. coli DSM 4243) with the aid of SstI and PvuII.
5. Part, with regulatory activity, of the stilbene synthase gene according to Claims 1 to 4.
6. Structural gene of the stilbene synthase gene according to Claims 1 to 4.
7. DNA containing the following sequence, which can be present either with or without intron, and the DNA sequences derived therefrom with stilbene synthase activity:

   

   

   
8. DNA consisting of or recombinant DNA containing the DNA sequence of the encoding DNA region according to Claim 7, including the DNA sequences derived therefrom with stilbene synthase activity.
9. Stilbene synthase gene or gene unit containing the DNA according to Claim 7.
10. Recombinant prokaryotic or eukaryotic DNA, which contains one or more stilbene synthase genes or gene units and/or their structural genes or their regulatory gene parts and/or the DNA according to Claims 1 to 9 as "foreign" DNA or as "additional" DNA.
11. Recombinant DNA according to Claim 10, which is contained in plant cells (including protoplasts) or plants (including parts of plants and seeds).
12. Vectors, which contain one or more stilbene synthase genes or gene units or their structural genes or their regulatory gene parts and/or the DNA according to Claims 1 to 9 and/or the recombinant DNA according to Claim 10.
13. Vector plasmid pGS 828.1 (obtainable from E. coli DSM 4243).
14. Transformed microorganisms, which contain one or more stilbene synthase genes or gene units and/or their structural genes or their regulatory gene parts and/or the DNA according to Claims 1 to 9 and/or the recombinant DNA according to Claim 10.
15. Escherichia coli strain Nurdug 2010 (corresponding to E. coli DSM 4243) and its mutants and variants.
16. Use of the stilbene synthase gene or the gene unit and/or its structural gene or its regulatory gene part and/or the DNA and/or the recombinant DNA and/or the vectors and/or the transformed microorganisms according to Claims 1 to 15 for the transformation of plant cells (including protoplasts) and plants (including parts of plants and seeds).
17. Transformed plant cells (including protoplasts) and plants (including parts of plants and seeds), which contain one or more stilbene synthase genes or gene units and/or their structural genes or their regulatory gene parts and/or the DNA and/or the recombinant DNA according to Claims 1 to 10 as "foreign" or "additional" DNA.
18. Process for the preparation of transformed plant cells (including protoplasts) and plants (including parts of plants and seeds) according to Claim 17, characterised in that

    (a) one or more stilbene synthase genes or gene units and/or their structural genes or their regulatory gene parts and/or the DNA and/or the recombinant DNA according to Claims 1 to 10 are introduced into the genome of plant cells (including protoplasts), and, if appropriate,

    (b) complete transformed plants are regenerated from the transformed plant cells (including protoplasts), and, if appropriate,

    (c) the desired parts of the plants (including seeds) are recovered from the transformed plants thus obtained.
19. Use of transformed plant cells (including protoplasts) according to Claim 17 for the production of propagation material and for the production of novel plants and propagation material thereof.
20. Propagation material, which can be obtained by the propagation of the transformed plant cells and plants according to Claim 17.
21. Transformed plant cells (including protoplasts) and plants (including parts of plants and seeds) according to Claim 17, the process for their preparation according to Claim 18, and their use according to Claim 19, and also the propagation material according to Claim 20, in which the plant cells and plants are tobacco, lucerne or potato plant cells and tobacco, lucerne or potato plants.

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